vitamin-k-semiquinone-radical and Osteosarcoma

Vitamin K (VK) is a group of fat‑soluble vitamins, which serve important roles in blood coagulation and bone metabolism. A recent study reported that several VK subtypes possess antitumor properties, however the antitumor effects of VK in osteosarcoma are unknown. The present study aimed to identify the antitumor effects of VK in osteosarcoma and the possible underlying mechanism of action. The effect of VK4 on cell viability was determined using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT) assay. Cellular and nuclear morphological changes were observed by phase contrast microscopy. Cell cycle analysis, apoptotic rate, mitochondrial membrane potential and levels of reactive oxygen species (ROS) were detected by flow cytometry. In vitro cancer cell migration activities were evaluated using a Wound healing assay and Transwell microplates. The results demonstrated that VK4 arrested the cells in S phase and induced apoptosis. Additional mechanistic studies indicated that the induction of apoptosis by VK4 was associated with the increased production of reactive oxygen species, dissipation of the mitochondrial membrane potential, decreased Bcl‑2 family protein expression levels and activation of caspase‑3. In conclusion, the results suggest that the sensitivity of U2OS osteosarcoma cells to VK4 may be as a result of mitochondrial dysfunction. As it is readily available for human consumption, VK4 may therefore present a novel therapeutic candidate for the treatment of patients with osteosarcoma.

Topics: Antineoplastic Agents; Apoptosis; Bone Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Humans; Membrane Potential, Mitochondrial; Mitochondria; Osteosarcoma; Vitamin K; Vitamins

The effect of the proinflammatory cytokine interleukin (IL)-1beta on the cellular proliferation of human osteoblastic cells (SaM-1) and osteosarcoma-derived cells (SaOS-2, HOS, and MG-63) was examined. IL-1beta stimulated the proliferation of SaM-1 and MG-63 cells, but had no effect on that of SaOS-2 or HOS cells. Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, the mRNA expression of IL-1 receptor type I (IL-1R1) was detected in SaM-1 and MG-63 cells consistently, but not in SaOS-2 or HOS cells in the proliferative stage. Neither the decoy inhibitory IL-1 receptor type II (IL-1R2) nor IL-1R antagonist mRNA was detected in any of the cell lines, suggesting that IL-1beta stimulated proliferation via IL-1R1. The IL-1beta -stimulated proliferation was inhibited by the MAPK kinase (MEK) inhibitor PD98059 but not by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 or the cyclooxygenase-2 specific inhibitor NS-398, suggesting that IL-1beta stimulated proliferation via MEK, without affecting prostaglandin E(2) synthesis. IL-1beta stimulated cellular proliferation but inhibited the synthesis of osteocalcin containing gamma-carboxylated glutamic acid (Gla-OSCAL). Both the increased proliferation and decreased Gla-OSCAL synthesis were suppressed by vitamin K(2) (VK(2)), which is a cofactor for gamma-carboxylase. Furthermore, the inhibitory effect of VK(2) on IL-1beta -stimulated proliferation was suppressed by warfarin. However, rifampicin the nuclear receptor steroid and xenobiotic receptor (SXR) ligand had no effect of IL-beta, suggesting that IL-1beta is involved in VK(2) dependent gamma-calboxylation but not SXR-activation. These results suggest that IL-1beta stimulated cellular proliferation via MEK and inhibited Gla-OSCAL synthesis, which were both inhibited by VK(2) via gamma-carboxylation.

Topics: Adult; Anticoagulants; Bone Neoplasms; Cell Line; Cell Proliferation; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Dinoprostone; Enzyme Inhibitors; Flavonoids; Glutamic Acid; Humans; Imidazoles; Interleukin-1; Interleukin-1beta; Male; Mitogen-Activated Protein Kinase Kinases; Nitrobenzenes; Osteoblasts; Osteocalcin; Osteosarcoma; p38 Mitogen-Activated Protein Kinases; Pregnane X Receptor; Protein Kinase Inhibitors; Pyridines; Receptors, Steroid; Reverse Transcriptase Polymerase Chain Reaction; Rifampin; RNA, Messenger; Sulfonamides; Vitamin K; Vitamins; Warfarin; Young Adult

The effect of insulin-like growth factor-I (IGF-I) and 2-methyl-3-all-trans-tetraphenyl-1,4-naphtoquinone (vitamin K2) on the synthesis of osteocalcin containing gamma-carboxyglutamic acid (Gla) residues which is the physiologically relevant form in bone metabolism was studied in cultured human osteoblast-like (MG-63) cells. Both IGF-I and vitamin K2 stimulated 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced osteocalcin containing Gla secretion in a concentration-dependent manner. This stimulatory effect of IGF-I and vitamin K2 was additive. Vitamin K2-enhanced osteocalcin containing Gla secretion was selectively suppressed by 3-(alpha-acetonyl-benzyl)-4-hydroxy-coumarin (warfarin). The stimulatory effect of IGF-I was completely abolished by the presence of cycloheximide; in contrast the effect of vitamin K2 was still observed in the presence of cycloheximide. Treatment of MG-63 cells with IGF-I caused an approximately 2.2-fold increase in osteocalcin mRNA levels (determined by reverse transcription-polymerase chain reaction). Vitamin K2 had no effect on either the stimulation of mRNA level by IGF-I or the basal level. IGF-I-stimulated osteocalcin containing Gla secretion was inhibited by one of its binding proteins (insulin-like growth factor binding protein-4) in a concentration-dependent manner. These findings suggest that the modes of action of IGF-I and vitamin K2 on 1.25(OH)2D3-induced osteocalcin containing Gla secretion in MG-63 cells are different.

Topics: 1-Carboxyglutamic Acid; Cycloheximide; Humans; Insulin-Like Growth Factor Binding Protein 4; Insulin-Like Growth Factor I; Osteoblasts; Osteocalcin; Osteosarcoma; Protein Synthesis Inhibitors; RNA, Messenger; Stimulation, Chemical; Tumor Cells, Cultured; Vitamin K; Warfarin

A human osteosarcoma cell line, HOS TE85 cells, and a mouse osteoblastic cell line, MC3T3-E1 cells, were cultured for 3 days in a medium containing various concentrations of menaquinone-4 (vitamin K2). As a result, the proliferation of HOS cells was suppressed by vitamin K2 in a dose dependent manner up to 56% of control by 10(-7)M of vitamin K2 and that of MC3T3-E1 cells was suppressed to 84% of control by 10(-6)M of vitamin K2. Vitamin K2 increased alkaline phosphatase activity in both kinds of cells. Warfarin counteracted the effect of vitamin K2 on osteoblastic cell proliferation. Our results show that vitamin K2 modulates proliferation and function of osteoblastic cells by some mechanisms including gamma-carboxylation system.

Topics: Animals; Cell Division; Dose-Response Relationship, Drug; Humans; Mice; Osteoblasts; Osteosarcoma; Tumor Cells, Cultured; Vitamin K; Vitamin K 2; Warfarin

An osteoblast-like human osteosarcoma cell line (U2-OS) has been shown to possess a vitamin K-dependent carboxylation system which is similar to the system in human HepG2 cells and in liver and lung from the rat. In an 'in vitro' system prepared from these cells, vitamin K1 was shown to overcome warfarin inhibition of gamma-carboxylation carried out by the vitamin K-dependent carboxylase. The data suggest that osteoblasts, the cells involved in synthesis of vitamin K-dependent proteins in bone, can use vitamin K1 as an antidote to warfarin poisoning if enough vitamin K1 can accumulate in the tissue. Five precursors of vitamin K-dependent proteins were identified in osteosarcoma and HepG2 cells respectively. In microsomes (microsomal fractions) from the osteosarcoma cells these precursors revealed apparent molecular masses of 85, 78, 56, 35 and 31 kDa. When osteosarcoma cells were cultured in the presence of warfarin, vitamin K-dependent 14C-labelling of the 78 kDa precursor was enhanced. Selective 14C-labelling of one precursor was also demonstrated in microsomes from HepG2 cells and from rat lung after warfarin treatment. In HepG2 cells this precursor was identified as the precursor of (clotting) Factor X. This unique 14C-labelling pattern of precursors of vitamin K-dependent proteins in microsomes from different cells and tissues reflects a new mechanism underlying the action of warfarin.

Topics: Antidotes; Carbon-Carbon Ligases; Electrophoresis, Polyacrylamide Gel; Factor X; Humans; Immunoblotting; Ligases; Lung; Microsomes; Microsomes, Liver; Molecular Weight; Osteoblasts; Osteosarcoma; Protein Precursors; Prothrombin; Tumor Cells, Cultured; Vitamin K; Warfarin

The recycling of vitamin K in the liver occurs via one or two dithiol-dependent reductases, which are strongly inhibited by coumarin derivatives such as warfarin. This inhibition may be partly overcome by the action of a NADH-dependent reductase, which is relatively insensitive for warfarin. In this paper we demonstrate that the osteoblast-like osteosarcoma UMR 106 does not contain the NADH-dependent reductase. Assuming that a similar enzyme distribution occurs in normal osteoblasts this explains the observation of Price and Kaneda, that the administration of vitamin K to rats efficiently counteracted the effect of warfarin on blood coagulation, but that the vitamin had no effect on the Gla-content of serum osteocalcin.

Topics: Animals; Blood Coagulation; Carboxy-Lyases; Liver; Microsomes; Osteosarcoma; Oxidoreductases; Rats; Rats, Inbred BN; Tumor Cells, Cultured; Vitamin K; Warfarin

Osteosarcoma cells grown in normal culture medium secrete bone gamma-carboxyglutamic acid protein (BGP, osteocalcin) which is identical with BGP purified from the bone matrix. Two tests indicate that the secreted medium protein contains the full complement of three gamma-carboxyglutamate residues present on BGP purified from the bone matrix. First, the secreted protein from ROS 17/2 and bone matrix BGP have identical isoelectric points (pI = 4.0). Second, they have identical hydroxyapatite binding behavior. If warfarin is added to the culture medium, the secreted protein has a higher isoelectric point (pI = 4.6) and a lower affinity for hydroxyapatite characteristic of thermally decarboxylated or non-gamma-carboxylated BGP. The observed shift in isoelectric point of secreted BGP after warfarin treatment from pH 4.0 to 4.6 is also reflected in the presence of pI = 4.1 and pI = 4.6 species intracellularly. These isoelectric species correspond to fully carboxylated BGP and noncarboxylated BGP, which are in the process of secretion. Addition of 10 micrograms/ml of warfarin causes a specific 47% reduction in secretion rate of BGP, while at the same time, the intracellular BGP concentration increases 3-fold. These phenomena appear related to the interruption by warfarin of the normal sequence of processing of precursor BGP proteins, as a new, immunoreactive species with a higher isoelectric pH not present in control cells appears to be responsible for the increased intracellular antigen within warfarin-treated cells. Our results show that vitamin K-dependent processing is important for normal secretion of BGP from the cell.

Topics: Animals; Calcium-Binding Proteins; Cells, Cultured; Chromatography, Gel; Isoelectric Focusing; Osteocalcin; Osteosarcoma; Rats; Vitamin K; Warfarin

Rat osteosarcoma cells respond to 1,25-dihydroxyvitamin D3 with a 6- to 10-fold increase in the secretion of citric acid. The time required to attain a half-maximal response is 12 h, a time course which is consistent with the postulated steroidal hormone action of this vitamin D metabolite. The citrate response is achieved by physiological concentrations of 1,25-dihydroxyvitamin D3, with half of the maximal response at a vitamin concentration of 0.03 ng/ml. Both the time course and the dose dependence of the citrate response closely parallel the previously reported stimulation of bone Gla protein synthesis by 1,25-dihydroxyvitamin D3 in these cells. Citrate and bone Gla protein bind avidly to bone mineral and are numerically the most abundant organic acid and protein in bone. The parallel secretion of both in 1,25-dihydroxyvitamin D3-treated osteoblastic cells suggests that they may act in tandem to mediate an action of this vitamin D metabolite on the mineral phase of bone.

Topics: Animals; Calcitriol; Calcium-Binding Proteins; Cell Line; Citrates; Dose-Response Relationship, Drug; Kinetics; Osteocalcin; Osteosarcoma; Rats; Vitamin K

Rat osteosarcoma cells respond to 1,25-dihydroxyvitamin D3 with a 6-fold increase in intracellular and secreted levels of the vitamin K-dependent protein of bone (BGP). The rise in intracellular BGP levels is half-maximal at 6.6 h and precedes the rise in medium BGP levels by 6 h, a time course which is consistent with the postulated steroid hormone action of 1,25-dihydroxyvitamin D3. This effect is achieved by physiological levels of 1,25-dihydroxyvitamin D3, with half of the maximal response at a vitamin concentration of 0.04 ng/ml. The specificity of this effect for BGP is demonstrated by the absence of a 1,25-dihydroxyvitamin D3 effect on total protein synthesis by these cells. To our knowledge, BGP is the first example of a bone protein whose rate of synthesis is dramatically and specifically increased by physiological levels of 1,25-dihydroxyvitamin D3. The possible functions of BGP in the biological actions of 1,25-dihydroxyvitamin D3 on bone are discussed.

Topics: 1-Carboxyglutamic Acid; Animals; Bone and Bones; Calcitriol; Cell Line; Dihydroxycholecalciferols; Glutamates; Hydroxycholecalciferols; Kinetics; Neoplasms, Experimental; Osteocalcin; Osteosarcoma; Protein Biosynthesis; Rats; Vitamin K